Fig. 2

IgSF11 signaling complexes induce PKM2 phosphorylation. a Comparative expression of PKM isoform 1 and isoform 2 in wild-type cells. Total RNA was isolated from BMMs and osteoclasts and analyzed by Q-PCR. The data are presented as the means ± S.D. b Expression of PKM isoform 1 and isoform 2 in BMMs and osteoclasts. Adult mouse brains and hearts were used as controls. Whole cell/tissue lysates were used for western blotting with the indicated antibodies. Coomassie blue staining is shown as the protein loading control. c Phosphorylation of PKM2 during osteoclast differentiation. BMMs were cultured with M-CSF + RANKL for the indicated days, and whole cell lysates were used for western blotting with the indicated antibodies. d Coimmunoprecipitation of IgSF11 with PKM2. IgSF11^−/− BMMs were retrovirally transduced with the indicated vectors, lysed and immunoprecipitated with anti- Flag antibodies, and western blotting was performed with the indicated antibodies. e IgSF11 induces the phosphorylation of c-Src, Fyn and HcK. IgSF11^−/− BMMs were retrovirally transduced with the indicated vectors, cultured with M-CSF + RANKL for two days and then stimulated with anti-hCD3 antibodies for the indicated times. Western blotting was performed with the indicated antibodies. f Src family kinase inhibitors attenuated PKM2 phosphorylation. IgSF11^−/− BMMs were retrovirally transduced with hCD3-iFL, cultured with M-CSF + RANKL for two days and then stimulated with anti-hCD3 antibodies for the indicated times in the presence or absence of inhibitors (PP2; 4 μmol·L^–1, bosutinib; 1 μmol·L^–1, dasatinib; 1 μmol·L^–1). Western blotting was performed with the indicated antibodies. The results are representative of at least three independent experiments